We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Proteomics Method Development. The implementation of label-free approaches for quantitative proteomics studies has been implemented. This methodology involves data-independent analyses (MSe) and an online UPLC method suitable for the analyses of proteins from complex samples. MSe can rapidly acquire accurate mass precursor and fragment ion information while simultaneously obtaining fairly accurate quantitative profiles from every detectable component in the sample, in theory. A comparison of the results obtained from depleted serum (depletion of the 14 most abundant proteins) followed by six different treatments is being compare. The results obtained by data-independent analyses (MSe) and be data-dependent analyses (DDA) are being evaluated. 2. Myositis Study. A differential proteomics project comparing the quantitation of proteins from sera of healthy individuals to individuals diagnosed with a rheumatic disease is underway. This work is in collaboration with F. Miller (EAG) as part of the EAGs study of families with twins or siblings discordant for systemic rheumatic disorders. The goal of this project is to determine whether a protein(s) can be identified that would allow for the early diagnosis, prognosis, and treatment of these diseases. Sera samples have been depleted and processed and are awaiting LC/MSe analysis. 3. Tissue Study. A protein expression study of LCM tissue is underway. Additionally, brain tissue has been evaluated for protein extraction. Initial experiments using brain tissue of rat resulted in the identity of about 300 proteins. A study comparing the extraction efficacies of proteins from frozen tissues versus heat stabilized tissue is under investigation. 4. COPD Study. A quantitative serum proteomics study of serum from healthy individuals versus individuals with varying degrees of COPD is underway. ITIH4 (inter-alpha-trypsin inhibitor heavy chain H4) is perhaps a biomarker. Using 2D LC/MSe the potential for ITIH4 as a biomarker has been investigated. It appears that ITIH4 is probably not a potential biomarker but after interrogating the MS data, the abundance of the protein LRG1 correlates with disease and, therefore, may be a biomarker for this disease. 5. Acrylamide Study. The effects of different sterilization parameters on rodent feed have been determined by measuring the amount of acrylamide that is produced in the feed with different sterilization parameters, primarily different steam autoclaving times. 6. Isoprostane Study. The generation of the most often measured isoprostane, 8-iso-PGF2&#945; by prostaglandin-endoperoxide synthase and arachidonic acid even with low enzymatic activity has been investigated. Isoprostanes are a family of compounds that are identical to their corresponding prostaglandins except for the difference in stereochemistry around the carbon-carbon bond at position eight. 7. Lipid/Fatty Acid Studies. A) Cholesterol and the metabolites of cholesterol in human serum are in the process of being measured. From initial studies, a correlation between the levels of these compounds and individuals with Acute Respiratory Distress Syndrome Treatment could be determined. Additional samples are currently being interrogated. B) The hypothesis that the autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis is a molecular target for lowering intraocular pressure (IOP) in animal models of glaucoma. Using ATX inhibitors, the role of autotaxin in regulation of intraocular pressure is characterized with the detection of LPA and LPC in the aqueous humor of eye. C) The role of COX-2 as an upstream regulator of p53 levels was investigated. DNA damage-induced increase in p53 protein levels was greatly reduced in COX-2 knockout (KO) mouse embryonic fibroblasts (MEFs) compared to that in wild type or COX-1 KO MEFs. Furthermore, a COX-2 specific inhibitor suppressed doxorubicin-induced p53 accumulation and cell death. D) The role of the retinoid acid-related orphan receptor gamma, ROR gamma, in the transcriptional regulation of lipid metabolic genes was determined by the quantitation of fatty acids. 8. Eicosanoid Studies. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry.